The overall goal of this application is to develop a scalable production system for the manufacture of an adeno- associated virus (AAV) serotype 8-based gene therapy vector bearing a human low density lipoprotein receptor (hLDLR) transgene for the treatment of familial hypercholesterolemia (FH). Recombinant AAV8 vector is one of a group of new generation vectors exclusively licensed to ReGenX with superior tissue transduction capabilities compared to currently available AAV vectors. AAV8 vectors efficiently target the liver and are therefore promising therapeutics for the treatment of dyslipidemias such as FH. This autosomal dominant disorder results from a deficiency of LDL receptor and is refractory to traditional pharmacologic therapy. In murine models of atherosclerosis, AAV8-hLDLR vectors are able to transduce up to 85% of hepatocytes, correct the underlying metabolic defect and prevent the resulting atherosclerosis. A major barrier to the translation of this research to the clinic has been the ability to produce AAV8 vector in sufficient quantities. The scalable process proposed here for the production of an AAV8-hLDLR vector will be based on the adenovirus-AAV hybrid (Ad hybrid) system initially developed for AAV2. The system relies entirely on adenovirus-AAV hybrid infection to deliver the recombinant AAV genome to packaging cell lines which contain the AAV capsid gene and is therefore suitable for use with large scale suspension cultures. We aim to generate Ad hybrid system components including AAV8 packaging cell lines and hLDLR Ad hybrids and use them to produce AAV8-hLDLR vector with acceptable yields and product potency. A novel modification in which the dual adenovirus/Ad hybrid infection process is replaced with a single infection will also be investigated. High throughput precise assays to characterize vector yield and potency will be specifically developed for use with the Ad hybrid system. In the final stages of Phase I the newly derived packaging lines will be adapted to suspension culture under serum free conditions and vector yields and potency assessed following hLDLR Ad hybrid infection. By the conclusion of phase I we aim to have established a high yielding suspension culture system for the production of AAV8-hLDLR which is both amenable to scale up and acceptable to regulatory agencies. These accomplishments will lead us to Phase II where optimal system components will be re-derived at a cGMP facility. In Phase II we envisage bioreactor-based optimization of the upstream process, development of a scalable downstream process and incorporation of several new in-process and QC assays. PUBLIC HEALTH RELEVANCE: Gene therapy targeted to the liver has the potential to treat many diseases of serious public health concern, including Hepatitis B and C, and inherited diseases such as hemophilia and familial hypercholesterolemia, the latter of which is characterized by high cholesterol levels and premature atherosclerosis. The overall goal of this project is to develop a large-scale production system for the commercial manufacture of a novel therapeutic for familial hypercholesterolemia.